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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Mol Microbiol. 2020 Aug 31;114(6):966–978. doi: 10.1111/mmi.14587

Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation

Thao T Truong 1, Andrea Vettiger 1, Thomas G Bernhardt 1,2,*
PMCID: PMC7775348  NIHMSID: NIHMS1630017  PMID: 32866331

Abstract

A peptidoglycan (PG) cell wall composed of glycans crosslinked by short peptides surrounds most bacteria and protects them against osmotic rupture. In Escherichia coli, cell elongation requires crosslink cleavage by PG endopeptidases to make space for the incorporation of new PG material throughout the cell cylinder. Cell division, on the other hand, requires the localized synthesis and remodeling of new PG at midcell by the divisome. Little is known about the factors that modulate transitions between these two modes of PG biogenesis. In a transposon-insertion sequencing screen to identify mutants synthetically lethal with a defect in the division protein FtsP, we discovered that mutants impaired for cell division are sensitive to elevated activity of the endopeptidases. Increased endopeptidase activity in these cells was shown to interfere with the assembly of mature divisomes, and conversely, inactivation of MepS was found to suppress the lethality of mutations in essential division genes. Overall, our results are consistent with a model in which the cell elongation and division systems are in competition with one another and that control of PG endopeptidase activity represents an important point of regulation influencing the transition from elongation to the division mode of PG biogenesis.

Keywords: cell division, elongation, peptidoglycan, cell wall, morphogenesis, cytokinesis

INTRODUCTION

The cell wall layer confers integrity and shape to most bacterial cells. It is composed of the heteropolymer peptidoglycan (PG), which forms a continuous meshwork of glycan chains crosslinked by covalently attached peptides {Holtje:1998gp}. Because this so-called PG sacculus surrounds the cytoplasmic membrane, its expansion and remodeling is intimately tied to the processes of growth and division (Typas et al., 2012; Zhao et al., 2017). In rod-shaped cells, the cylindrical portion of the PG matrix must first be elongated before a localized synthesis of new PG at midcell is initiated to promote cell division. Despite their importance for growth and morphogenesis, the factors that modulate the transition between the elongation and division modes of PG biogenesis remain incompletely described.

Two major types of cell wall synthases participate in PG matrix assembly during cell elongation and division. The class A penicillin-binding proteins (aPBPs) are bifunctional and possess both PG glycosyltransferase (PGTase) activity to polymerize the PG glycan strands and transpeptidase (TPase) activity to form crosslinks between the peptide stems (Sauvage et al., 2008). The SEDS proteins also have PGTase activity (Meeske et al., 2016), and they have been recently shown to join with TPases called class B PBPs (bPBPs) in forming a second type of bifunctional PG synthase (Rohs et al., 2018; Taguchi et al., 2019; Sjodt et al., 2020).

In Escherichia coli and other rod-shaped bacteria, cell elongation is carried out by the Rod system (elongasome) (Typas et al., 2012; Zhao et al., 2017). The essential PG synthase in this system is formed by a SEDS-bPBP complex called RodA-PBP2 (Meeske et al., 2016; Cho et al., 2016; Rohs et al., 2018). In association with filaments of the actin-like protein MreB and other membrane components of the Rod system, RodA-PBP2 is thought to insert new hoops of PG at dispersed sites throughout the cell cylinder to promote its elongation (Domínguez-Escobar et al., 2011; Garner et al., 2011; van Teeffelen et al., 2011). The aPBPs are also required for proper expansion of the wall during elongation, but their precise role remains to be determined. An attractive possibility is that these enzymes fortify the growing cylinder by adding PG material to a framework laid down by the Rod system (Cho et al., 2016; Vigouroux et al., 2020). In addition to PG synthases, elongation of the sacculus requires bonds in the PG network to be broken to make space for the insertion of new material. In E. coli, the space-making enzymes are the PG endopeptidases MepS and MepM, at least one of which is essential for growth in rich medium (Singh et al., 2012).

After a period of elongation, cell division is initiated by the formation of the cytokinetic ring (septal ring, divisome) at midcell (Du and Lutkenhaus, 2017). This structure is underpinned by treadmilling polymers of the tubulin-like protein FtsZ (Bi and Lutkenhaus, 1991; Loose and Mitchison, 2013; Bisson-Filho et al., 2017; Xinxing Yang et al., 2017), which together with a collection of FtsZ-binding proteins like FtsA, ZipA, and ZapA comprises the Z-ring (Lutkenhaus et al., 2012). This dynamic cytoskeletal structure promotes the recruitment of dozens of proteins to the division site to assemble the mature divisome capable of promoting cell constriction and the synthesis of the multilayered septal PG material that will eventually form the daughter cell poles (Du and Lutkenhaus, 2017). The essential PG synthase of the divisome is a SEDS-bPBP complex formed by FtsW and PBP3 (FtsI) (Taguchi et al., 2019). In E. coli, the aPBP PBP1b is also thought to be involved in septal PG biogenesis (Bertsche et al., 2006; Müller et al., 2007; Typas et al., 2012), and similar to cell elongation, may fortify a foundational structure deposited by FtsWI.

Septal PG synthesis by the divisome is thought to be triggered and maintained by a self-enhancing process called the septal PG loop (Liu et al., 2019), in which the bitopic transmembrane protein FtsN is thought to play a key role (Gerding et al., 2009) (Fig. 1). According to this model (Gerding et al., 2009), FtsN is initially recruited to the division site through an interaction between its N-terminal cytoplasmic domain and FtsA (Busiek et al., 2012). This interaction is then thought to provide two signals for the activation of PG synthesis by a subset of the synthases in the divisome, one via FtsA in which the FtsEX ABC-complex has also been implicated (Du et al., 2016), and another via the essential peptide within the FtsN periplasmic domain (Gerding et al., 2009; Liu et al., 2015). Genetic evidence from hyperactive division mutants suggests that these signals are likely to act directly or indirectly on the conserved subcomplex of FtsQ, FtsL, and FtsB (FtsQLB) (Liu et al., 2015; Tsang and Bernhardt, 2015), which in-turn are thought to communicate with FtsWI to activate PG synthesis. The septal PG produced is processed by amidase enzymes involved in cell separation to generate peptide-free (denuded) PG glycans (Heidrich et al., 2001). The periplasmic SPOR domain of FtsN binds these products (Ji-Chun Yang et al., 2004; Müller et al., 2007; Gerding et al., 2009; Arends et al., 2010; Yahashiri et al., 2017), thus recruiting more FtsN to the division site, which stimulates more septal PG synthesis and processing, promoting more FtsN recruitment, and so on in a reinforcing cycle that drives the formation of the new daughter cell poles (Gerding et al., 2009) (Fig. 1).

Figure 1. The activation loop promoting septal PG biogenesis.

Figure 1.

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Shown is a schematic summarizing the current model for the activation of septal PG biogenesis. Several divisome proteins that are also implicated in the pathway such as the FtsEX ABC complex and FtsK are omitted for simplicity. See text for details.

The initial goal of this study was to investigate the function of the auxiliary divisome protein FtsP, a member of the multicopper oxidase superfamily that lacks key catalytic residues (Reddy, 2007; Samaluru et al., 2007; Tarry et al., 2009). FtsP is transported to the periplasm by the Tat system (Stanley et al., 2000) and is a late recruit to the divisome (Tarry et al., 2009). Like FtsN (Dai et al., 1993), FtsP overproduction has been found to suppress a variety of division defects (Reddy, 2007; Samaluru et al., 2007), but its role in cell division has remained mysterious. To better understand its function, we screened for mutants synthetically lethal with an FtsP defect using transposon sequencing (Tn-Seq) (van Opijnen and Camilli, 2013). In addition to providing evidence supporting a role for FtsP in the septal PG loop, our analysis revealed that mutants inactivated for FtsP or other auxiliary divisome components involved in the activation of septal PG biogenesis are highly sensitive to elevated activity of the elongation endopeptidases MepS or MepM. Several lines of investigation indicate that increased endopeptidase activity in these cells interferes with divisome maturation and the initiation of the septal PG loop. Overall, our results are consistent with a model in which the cell elongation and division systems are in competition with one another and that control of PG endopeptidase activity represents an important regulatory point influencing the transition from elongation to the division mode of PG biogenesis.

RESULTS

Identification of mutants synthetically lethal with an FtsP defect

In an effort to better understand FtsP function, we used Tn-Seq (van Opijnen and Camilli, 2013) to identify mutations causing a synthetical lethal phenotype when combined with an ftsP deletion. Transposon mutagenized libraries of wild-type and a ΔftsP strain of E. coli were therefore prepared using a conjugation-based transposon delivery system. To maximize the chances of identifying synthetic lethal partners for ftsP, the libraries were propagated on LB agar without NaCl (LB0N), a growth condition known to be less permissive for ftsP and other division mutants (Reddy, 2007; Samaluru et al., 2007). Following growth on LB0N, genomic DNA was isolated from each library and the transposon insertion profiles were analyzed by Tn-Seq. Comparison of the insertion profiles between the strains led to the identification of a number of genes with a reduction in mapped transposon insertions in the ΔftsP mutant relative to wild-type (Table S1), including several with a direct or indirect connection to cell division or cell wall synthesis like: nlpI, ponB (mrcB), ftsN, ftsK, lon, glmU, and ldcA. Based on: (i) the magnitude of the depletion ratio (insertion reads in ΔftsP/wildtype < 0.35), (ii) the statistical significance of the profile difference (p-value < 0.05), and (iii) a visual inspection of the insertion profiles for candidate genes (Fig. 2 and Fig. S1), we chose nlpI, ponB (mrcB), and ftsN as the most attractive potential synthetic lethal partners of ftsP for further study.

Figure 2. Identification of synthetic lethal partners of ftsP.

Figure 2.

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Shown are transposon insertion profiles of the indicated genomic regions in wild-type and ΔftsP strains. Each vertical line represents a sequenced insertion site, and the heights of the lines correspond to the number of reads sequenced at those sites. Regions encoding the C-terminal SPOR domain or the essential domain of ftsN are highlighted in pink or purple, respectively.

The ponB gene encodes PBP1b, a major aPBP-type PG synthase. NlpI is an outer membrane lipoprotein that serves as an adaptor for the protease Prc and is required for the turnover of the elongation PG endopeptidase MepS (Singh et al., 2015). Note that transposon insertions in the prc gene were already depleted in the wild-type strain background, which prevented it from also being identified as a hit in the screen as expected based on the nlpI result. Because they are non-essential genes, the Tn-seq results for both nlpI and ponB were straightforward to interpret. Insertions were tolerated throughout the genes in the wild-type strain, but not in the ΔftsP mutant (Fig. 2). The results for FtsN were more complicated because it contains an essential peptide in its periplasmic domain in addition to the non-essential C-terminal SPOR domain (Ji-Chun Yang et al., 2004; Müller et al., 2007; Gerding et al., 2009; Arends et al., 2010; Yahashiri et al., 2017). Accordingly, transposon insertions in ftsN were tolerated downstream of the region encoding the essential peptide, which would result in the production of a truncated protein lacking the SPOR domain (Fig. 2). These insertions were no longer tolerated in the absence of FtsP, indicating that the SPOR domain has likely also become essential in this background (Fig. 2).

To validate the Tn-Seq results, we first attempted to inactivate the candidate synthetic lethal partner genes in the context of a ΔftsP strain. Selections for constructing the double deletion strain were performed on minimal medium, which is often a permissive condition for mutants with severe division defects (Bernhardt and de Boer, 2005; Goehring et al., 2007). Only the double ΔftsP ΔnlpI mutant yielded stable isolates with this procedure. Consistent with the Tn-seq results, this double mutant exhibited a severe plating defect on LB0N agar and grew poorly relative to wild-type and single mutant controls in LB0N liquid (Fig. 3A–B). Imaging of the cells revealed that the growth defect under these non-permissive conditions resulted from impaired division that led to cell elongation and some cell lysis (Fig. 3C–D).

Figure 3. The synthetic lethal phenotype of ΔnlpI ΔmepS cells depends on mepS.

Figure 3.

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A. Cells of DL81 [ΔftsP], TU135 [ΔnlpI], TT185 [ΔftsP ΔnlpI], and TT189 [ΔftsP ΔnlpI ΔmepS] were grown overnight in minimal M9 medium supplemented with 0.2% arabinose (M9 arabinose) at 30°C and normalized for cell density (OD600 = 1). The samples were then serially diluted (10−1 – 10−6) and 5 μL of each dilution was spotted on LB0N and M9 arabinose. Plates were photographed after overnight incubation at 37°C. B. Growth curves of the strains from (A). Overnight cultures were diluted to OD600 = 0.025 in M9 arabinose and grown to mid-log phase at 30°C. The cells were then back-diluted to OD600 = 0.1 in LB0N and grown at 37°C. The optical density of each strain was measured every 15 minutes for 2.5 hours. C. Samples of cells from (B) were removed at the 150 minute time-point, fixed, and imaged using phase contrast microscopy. Note that cells lacking MepS are wider than normal. Bar = 4 μm. D. The strains imaged in (D) were analyzed using Oufti to determine cell length (experiments were performed in triplicate, 200–300 cells were analyzed per strain in each replicate). The average length and standard deviation for each strain are indicated by the black lines. The cell lengths were compared using the Kruskal-Wallis test in combination with Dunn’s multiple comparisons test in GraphPad Prism 8.0 to determine significance. NS, difference not statistically significant. ****, difference significant at P < 0.0001.

The other two potential synthetic lethal partners were validated by inactivating the candidate gene in the context of an FtsP depletion strain. For ftsN, a deletion was constructed to remove the region coding for the non-essential SPOR domain (ΔSPORftsN). As expected from the Tn-Seq analysis, depletion of FtsP in the context of the ΔSPORftsN allele led to a growth defect on LB0N agar (Fig. 4A). Additionally, imaging indicated that depletion of FtsP enhanced the division problems caused by the ΔSPORftsN allele, leading to the formation of longer filamentous cells (Fig. 4B). Similarly, depletion of FtsP in cells deleted for ponB resulted in a severe growth defect on LB0N agar (Fig. 4C). However, rather than cell filamentation, the combined inactivation of FtsP and PBP1b caused cell lysis, which appeared to be preceded by the formation of blebs emanating from sites of division, a phenotype indicative of a catastrophic failure in the septation process (Fig. 4D).

Figure 4. Synthetic lethal phenotypes of ΔftsP cells inactivated for the SPOR domain of FtsN or PBP1b.

Figure 4.

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A. Cells of DL81(attλTT50) [ΔftsP (Para::ftsP)] and TT181(attλTT50) [ΔSPORftsN ΔftsP (Para::ftsP)] were grown overnight in M9 arabinose at 30°C and normalized for cell density (OD600 = 1). The samples were then serially diluted (10−1 – 10−6) and 5 μL of each dilution was spotted on LB0N with and without 0.2% arabinose and M9 arabinose. Plates were photographed after overnight incubation at 30°C. B. Overnight cultures of DL81(attλTT48) [ΔftsP (Para::ftsP)], TT154 [ΔSPORftsN], and TT179(attλTT48) [ΔftsP ΔSPORftsN (Para::ftsP)] were diluted 1:100 in M9 supplemented with 0.2% glucose (M9 glucose) and grown to mid-log phase at 30°C. They were then back-diluted to OD600 = 0.05 in LB0N and grown at 30°C for two hours. Cells were imaged using phase contrast microscopy. Bar = 4 μm. C. Cells of DL81(attλTT48) [ΔftsP (Para::ftsP)] and TT172(attλTT48) [ΔponB ΔftsP (Para::ftsP)] were grown, diluted, and spotted on agar as in panel A. Plates were photographed after overnight incubation at 37°C. Note that the Para::ftsP construct here differs from the one used in panel A. It includes the native ribosome-binding site (RBS) for ftsP, whereas the one in (A) uses the phi10 RBS from phage T7. D. Left: Overnight cultures of MM11 [Para::ponB] and TT151 [Para::ponB ΔftsP] were diluted 1:150 in LB0N and grown at 30°C to mid-log phase. Cells were imaged using phase contrast and fluorescence microscopy on a LB0N agarose pad containing 1 μg/ml propidium iodide (PI). Arrow points to a cell lysing from a septal lesion. Bar = 4 μm. Right: The fraction of lysed cells (PI positive) was quantified from multiple images. Data is represented as violin plots with median (solid black line) and quantiles (dotted lines) indicated. Dots represent the median from individual images (n =30) collected from three biological replicates. The total number of cells is indicated on graph. **** = p < 0.0001 unpaired t-test with Welch’s correction for unequal standard deviation. Note that depletion of PBP1b in a ΔftsP strain was lethal on LB0N just as FtsP depletion was lethal in a ΔponB background. See Figure S2.

Overall, the validated synthetic lethal relationships with FtsP suggest that it plays a role in PG biogenesis and that its function may be partially redundant with FtsN in the activation of the septal PG loop (see Discussion). This potential function for FtsP will be explored as part of future investigations. The remainder of this report will focus on the synthetic lethal phenotype of the double ΔftsP ΔnlpI mutant and how it led us to uncover an antagonistic relationship between the activity of cell elongation PG endopeptidases and cell division.

Elevated PG endopeptidase activity is lethal in mutants impaired for cell division

Given the role of NlpI in the turnover of MepS in the periplasm (Singh et al., 2015), we hypothesized that the synthetic lethal phenotype of the ΔftsP ΔnlpI mutant was related to elevated levels of MepS. Accordingly, deletion of mepS restored growth and division of ΔftsP ΔnlpI cells in LB0N medium (Fig. 3). To test whether elevated MepS activity alone is sufficient for the observed synthetic lethality, mepS was overexpressed from the IPTG-inducible tac promoter (Ptac) in cells with or without an ftsP deletion. Induction of mepS expression was well tolerated in FtsP+ cells, but induced a severe growth defect in ΔftsP cells on LB0N (Fig. 5). Similar to the double ΔftsP ΔnlpI mutant, this growth defect was accompanied by a block in cell division (Fig. 6). However, the filamentation was more pronounced and much less lysis was observed when MepS was overproduced in ΔftsP cells relative to the ΔftsP ΔnlpI mutant (Fig. 3B and 6B). This phenotypic difference most likely stems from the effect of NlpI inactivation on the stability and/or activity of proteins other than MepS (Banzhaf et al., 2020). Overproduction of a catalytically inactive version of MepS, MepS(C68A) (Singh et al., 2012), failed to induce a growth defect in either a wildtype or ΔftsP background, indicating that endopeptidase activity is required for the lethal effect of MepS overproduction on ΔftsP cells (Fig. 5).

Figure 5. Overproduction of MepS is sufficient to induce a growth defect in ΔftsP cells.

Figure 5.

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Derivatives of TB153 [ΔmepS] and TT164 [ΔmepS ΔftsP] harboring pGL66 [Ptac::mepS], pGL67 [Ptac::mepS(C68A)], and pGL70 [Ptac::empty] integrated at the attλ site on the chromosome were grown overnight in LB at 30°C. They were then serially diluted as in Figure 2 and spotted on LB0N agarose plates supplemented with the indicated concentrations of IPTG. Plates were photographed following overnight incubation at 37°C.

Figure 6. Overproduction of endopeptidases is lethal in several mutants defective for division.

Figure 6.

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A. Derivatives of TB28 [WT], DL81 [ΔftsP], TB44 [ΔenvC], and TT154 [ΔSPORftsN] harboring pGL66 [Ptac::mepS], pHC964 [Ptac::mepM], and pGL70 [Ptac::empty] integrated at the attλ site on the chromosome were grown overnight and processed as described in Figure 4. B. Representative phase-contrast images of strains from panel (A). Overnight cultures of each strain were grown in LB at 30°C, diluted 1:100 in LB0N, and grown to mid-log phase at 37°C. Cultures were then back-diluted to OD600 = 0.02 in LB0N with 50 μM IPTG and grown at 37°C. Cells were fixed at OD600 = 0.2–0.3 and imaged. Bar = 4 μm.

Given the genetic connections between FtsP and PG synthesis, we next investigated whether elevated endopeptidase activity was lethal to other division mutants impaired for the activation of septal PG biogenesis by the divisome. We therefore chose ΔSPORftsN and a deletion of envC for further analysis. EnvC is a non-essential division protein that activates the amidases AmiA and AmiB at the septum (Uehara et al., 2010). This activation promotes the septal PG loop by generating denuded glycan strands that bind the SPOR domain of FtsN and recruit it to the division site (Müller et al., 2007; Gerding et al., 2009; Arends et al., 2010; Yahashiri et al., 2015). Just as for the ΔftsP mutant, overproduction of MepS induced a lethal growth and division defect in both ΔenvC and ΔSPORftsN cells (Fig. 6). This effect was not specific for MepS as a similar phenotype was induced by overproduction of the unrelated MepM endopeptidase in all three division impaired mutants (Fig. 6). We therefore conclude that increased PG endopeptidase activity is antagonistic to cell division in genetic backgrounds defective in the activation of septal PG biogenesis.

Elevated MepS activity prevents proper septal ring activation in ΔftsP cells

To determine the stage of cell division affected by elevated PG endopeptidase activity in ΔftsP cells, the process was monitored using either ZapA-GFP as a proxy for Z-ring formation or GFP-FtsN for the completion of divisome assembly and divisome activation. For cells expressing ZapA-GFP, the frequency of normal Z-ring formation per cell area was quantified using the spot detector of the Fiji TrackMate plugin (see Experimental Procedures). MepS overproduction in wild-type cells led to a minor reduction in the frequency of normal Z-rings (Fig. 7A and Table 1). A similar effect was observed in the ΔftsP mutant, with the filamentous cells overexpressing mepS displaying roughly half the number of normal Z-rings relative to wild-type cells with the empty vector (Fig. 7A and Table 1). In addition to normal rings, these filaments also harbored a number of aberrant ZapA-GFP structures appearing as double rings or spirals, indicating potential problems in the formation of stable/condensed Z-rings. The effect of MepS overproduction on GFP-FtsN localization in ΔftsP cells was more severe. Cells overproducing MepS harbored approximately one third as many FtsN rings relative to wild-type cells with the empty vector, with some filaments displaying a substantial deficit in FtsN localization (Fig. 7B and Table 1). Given that proper FtsN recruitment requires the activation of septal PG biogenesis and the production of denuded glycan strands, we conclude from these results that MepS overproduction is likely to be interfering with divisome activation in cells lacking FtsP.

Figure 7. MepS overproduction interferes with proper septal ring maturation in ΔftsP cells.

Figure 7.

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A. Shown are representative phase-contrast (top row) and fluorescence (bottom row) micrographs showing the localization of ZapA-GFP in the indicated strains. Derivatives of NP1 [zapA-gfp] and TT236 [ΔftsP zapA-gfp] harboring pGL66 [Ptac::mepS] or pGL70 [Ptac::empty] integrated at the attλ site on the chromosome were grown overnight in LB at 30°C, diluted 1:100 in LB0N and grown to mid-log phase at 37°C. Cultures were then back-diluted to OD600 = 0.02 in LB0N with 50 μM IPTG and grown at 37°C to OD600 = 0.2 – 0.3 and imaged using phase-contrast and GFP optics. Arrowheads indicate examples of aberrant ZapA-GFP rings. Bar = 4 μm. B. Shown are representative phase-contrast (top row) and fluorescence (bottom row) micrographs showing the localization of GFP-FtsN in the indicated strains. Derivatives of TB28(attHKTU194) [WT (Para::gfp-ftsN)] and DL81(attHKTU194) [ΔftsP (Para:: gfp-ftsN)] harboring pGL66 [Ptac::mepS] or pGL70 [Ptac::empty] integrated at the attλ site on the chromosome were grown and imaged as in panel A. Bar = 4 μm.

Table 1.

Septal ring formation in cells overexpressing mepS.

Genotype Normal Z-rings/100μm2 Normal FtsN-rings/100μm2
WT Ptac::empty 19 ± 1.9 14 ± 1.8
WT Ptac::mepS 14 ± 2.3 10 ± 1.4
ΔftsP Ptac::empty 15 ± 1.4 15 ± 2.5
ΔftsP Ptac::mepS 10 ± 1.4 6 ± 1.5
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Images from Fig. 5A and 5B were analyzed using the Fiji TrackMate plugin to determine the number of normal Z- or FtsN rings in each image, respectively. 1000–1200μm2 of total cell area was analyzed for each strain for each of three biological replicates. Shown are the average number of normal rings per 100μm2 ± standard deviation.

A hyperactive ftsL allele suppresses the MepS-induced division defect

To further investigate the nature of the endopeptidase-induced division block, we tested whether or not it could be alleviated by mutations that enhance divisome activity. Indeed, the hyperactive division allele of ftsL, ftsL(E88K) (Liu et al., 2015; Tsang and Bernhardt, 2015), suppressed the synthetic lethality of ΔftsP ΔnlpI cells (Fig. 8A) and rescued ΔftsP, ΔenvC, and ΔSPORftsN cells from the lethality caused by MepS overproduction (Fig. 8B). The ftsL(E88K) allele and other similarly hyperactive mutants have previously been shown to bypass the requirement for FtsN and other division proteins that are normally thought to be essential for the activation of septal PG synthesis by FtsWI within the divisome (Liu et al., 2015; Tsang and Bernhardt, 2015; Liu et al., 2019). Because ftsL(E88K) similarly relieves the division block induced by MepS, we infer that elevated endopeptidase activity interferes with the activation of PG biogenesis at the division site.

Figure 8. The ftsL(E88K) allele suppresses the lethality of MepS overproduction in mutants impaired for cell division.

Figure 8.

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A. Cells of DL81 [ΔftsP], TU135 [ΔnlpI], MT10 [ftsL(E88K)], TT185 [ΔftsP ΔnlpI], and TT224 [ΔftsP ΔnlpI ftsL(E88K)], were grown overnight in M9 arabinose at 30°C, serially diluted, and then plated on LB0N and M9 arabinose as indicated. Plates were grown at 37°C overnight prior to imaging. The ftsL(E88K) allele is abbreviated as ftsL*. B. Cells of MT10 [ftsL (E88K)], DL81 [ΔftsP], TT234 [ΔftsP ftsL(E88K)], TB44 [ΔenvC], TT230 [ΔenvC ftsL(E88K)], TT154 [ftsNΔSPOR], and TT232 [ftsNΔSPOR ftsL (E88K)] with pGL66 [Ptac::mepS] or pGL70 [Ptac::empty] integrated at the attλ site were grown overnight in LB at 30°C, serially diluted, and plated on LB0N with the indicated concentrations of IPTG. Plates were grown at 37°C overnight prior to imaging.

Inactivation of MepS promotes cell division

Given that overproduction of MepS is detrimental to mutants impaired for cell division, we wondered whether the opposite might also be true. Would MepS inactivation promote cell division? To test this possibility, we took advantage of the conditional essentiality of the FtsEX system for cell division. This system promotes the activation of both septal PG synthesis and its processing by the amidases at the division site (Desirée C Yang et al., 2011; Du et al., 2016). It is normally essential for growth and division on LB0N, but is dispensable in minimal medium (Reddy, 2007; Desirée C Yang et al., 2011) (Fig. 9A–C). However, deletion of mepS was found to restore growth and partially rescue the cell division defect of ΔftsEX cells grown on LB0N (Fig. 9A–C). Similarly, inactivation of MepS also partially rescued the high-temperature growth defect of a mutant harboring a temperature-sensitive allele (ftsQ1) (Begg et al., 1980) of the essential ftsQ division gene (Fig. 9D). Deleting mepS not only rescues division defects, it was also found to be synthetically lethal with the hyperactive division allele ftsL(E88K) (Fig. 9E). Thus, just as overproduction of MepS antagonizes cell division, its inactivation appears to promote the process.

Figure 9. Inactivation of MepS suppresses division defects and is lethal when division is activated.

Figure 9.

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A. Cells of TB28 [WT], TB153 [ΔmepS], TU191(attλTU188) [ΔftsEX (Para::ftsEX)], and GL8(attλTU188) [(ΔftsEX ΔmepS (Para::ftsEX)] were grown overnight in M9 arabinose at 30°C, serially diluted, and plated on M9 arabinose or LB0N agar. Plates were incubated 37°C overnight and then photographed. Strains labeled ΔftsEX in the figure are ftsEX depletion strains. B. Shown are representative phase-contrast images of strains from (A). Overnight cultures were grown in M9 arabinose at 30°C then diluted to OD600 = 0.00075 in LB0N supplemented with 0.2% glucose. Cells were fixed at OD600 = 0.2 – 0.3 and imaged by phase-contrast microscopy. C. Images obtained as described in (B) were analyzed using Oufti to determine cell length, as in Figure 2D. D. Cells of TB28 [WT], TB153 [ΔmepS], TT198 [ftsQ1], and TT201 [ftsQ1 ΔmepS] were grown overnight in LB at 30°C, serially diluted, and then plated on LB agar. Plates were incubated at 30°C or 42°C overnight as indicated and then imaged. E. Cells of TB28 [WT], MT10 [ftsL(E88K)], TB153 [ΔmepS], and MT20 [ftsL(E88K) ΔmepS] were grown overnight in LB at 30°C, serially diluted, and then plated on LB agar and half-strength LB0N (0.5 X LB0N) agar as indicated. In this case, 0.5 X LB0N was used because the phenotypes of ftsL(E88K) and ΔmepS are more pronounced in this medium. Plates were incubated at 37°C overnight prior to imaging.

DISCUSSION

In order to gain a better understanding of the function of FtsP in cell division, we searched for genes that become essential when it is inactivated. Results from the screen along with associated follow-up experiments led to two new and related insights. The first is that FtsP is likely to be involved in the activation of septal PG biogenesis by the divisome in a manner analogous to FtsN. Secondly, we found that mutants impaired for this activation event are hypersensitive to elevated activity of the cell elongation endopeptidases. This latter finding provides experimental support for the long-standing idea in the field that cell elongation and cell division might be competing processes (Lleo et al., 1990; Begg et al., 1990).

A potential role for FtsP in the activation of septal PG biogenesis

FtsN was originally identified based on its ability to suppress the lethal division defect of a temperature sensitive allele of ftsA when overproduced (Dai et al., 1993). Overexpression of ftsN was then subsequently found to suppress the lethality of mutations in a variety of essential division genes (Dai et al., 1993; Geissler and Margolin, 2005; Reddy, 2007; Pichoff et al., 2018). It has been known for some time that the overproduction of FtsP also suppresses the lethal phenotypes of many of the same division mutants (Kato et al., 1988; Reddy, 2007; Samaluru et al., 2007; Pichoff et al., 2018). Our results further connect the activity of these two division proteins. The ponB gene encoding PBP1b was found to be a synthetic lethal partner with ftsP just as it was previously shown to become essential in slm117 cells producing a hypomorphic variant of FtsN (slm117FtsN) lacking the SPOR domain (Liu et al., 2015). The terminal phenotype of both ΔftsP ΔponB and slm117ftsN ΔponB cells was also found to be similar. In both cases the cells formed blebs at midcell and lysed, indicating a catastrophic defect in septal PG biogenesis. Cells deleted for ftsP or the SPOR domain-encoding portion of ftsN are also similarly hypersensitive to the overproduction of elongation endopeptidases. Thus, not only does the overproduction of FtsN and FtsP result in common suppressive phenotypes, their inactivation leads to similar division defects.

Taken together, the genetic results reported here and in past studies support a role for FtsP in the activation of septal PG biogenesis similar to that of FtsN. However, because ΔftsP and ΔSPORftsN were found to form a synthetic lethal pair, FtsP is likely to be participating in a pathway for septal PG synthesis activation that is parallel to FtsN rather than working with it in the same pathway. Recently, another SPOR domain protein called DedD with a similar domain architecture to FtsN was found to also contribute to the septal PG loop (Liu et al., 2019). Thus, although the essentiality of FtsN indicates that it plays the most critical role in stimulating septal PG synthesis relative to the non-essential DedD and FtsP proteins, there appear to be several inputs into the process. The challenge going forward will be to determine how these activation pathways are integrated by the divisome and to elucidate the molecular mechanism(s) by which they each promote septal PG synthesis.

Competition between elongation and division

Models for cell growth involving a competition between cell elongation and division have been proposed and discussed in the literature for many years (Lleo et al., 1990; Begg et al., 1990). However, experimental support for such a competition has only recently emerged. In a study about the rate-limiting steps of division, it was shown that cells harboring a temperature-sensitive allele of ftsI (ftsI23) elongate faster than a wild-type control when they were grown at the permissive temperature (Coltharp et al., 2016), suggesting that cell elongation is stimulated when division is impaired. Analogously, cells with a variant of PBP2 that hyper-activates the Rod system are longer than cells with wild-type PBP2 (Rohs et al., 2018), implying that division may occur less frequently when cell elongation is stimulated. Here, we provide further support for a competition between the two major growth modes and implicate endopeptidase activity in the antagonistic relationship.

Cells with ΔftsP, ΔenvC, or ΔSPORftsN mutations were shown to be hypersensitive to overproduction of the elongation endopeptidases MepS or MepM. Each of these mutants is defective in a factor that has been implicated in the septal PG loop, suggesting that elevated activity of enzymes involved in promoting cell elongation interferes with the activation of septal PG biogenesis by the divisome. Accordingly, the lethal division defect of these cells was suppressed by the ftsL(E88K) allele that is thought to be hyperactive in triggering septal PG biogenesis (Tsang and Bernhardt, 2015; Liu et al., 2019). Furthermore, ΔftsP cells overproducing MepS were found to be defective in the formation of GFP-FtsN rings, a phenotype expected in cells that cannot initiate the septal PG loop to generate the denuded PG glycans that robustly recruit FtsN to the division site via its SPOR domain (Gerding et al., 2009; Arends et al., 2010; Yahashiri et al., 2017). Antagonism of cell division by the elongation endopeptidases is further supported by the finding that inactivation of MepS suppresses the conditional lethality of mutations in ftsEX and ftsQ, which also encode factors implicated in triggering septal PG biogenesis (Tsang and Bernhardt, 2015; Du et al., 2016; Liu et al., 2019). Thus, our findings provide strong genetic evidence that cell elongation can be an impediment to division, and that the control of elongation endopeptidase activity is an important factor in balancing the competition between growth modes (Fig. 10).

Figure 10. Endopeptidase activity antagonizes cell division.

Figure 10.

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Model for the effect of endopeptidase activity on the processes of elongation and division. Crosslink-cleavage activity by the endopeptidases creates space to promote (indicated by plus sign) the insertion of new strands (red) in the existing PG meshwork (green), enabling cells to elongate. Our results indicate that although this activity promotes PG synthesis during cell elongation, it interferes with the activation of septal PG synthesis at the division site (indicated by the minus sign). See text for details.

Although the mechanistic basis for the competition between division and elongation remains to be determined, a likely source is the limited supply of the lipid II precursor used by the PG polymerases to synthesize the glycan strands. If true, impaired activation of septal PG biogenesis would prevent effective siphoning of substate to the divisome, causing it to be more sensitive to competition for precursor from other PG synthetic systems. Endopeptidase overproduction has previously been shown to enhance PG synthesis by the aPBPs (Lai et al., 2017). Thus, one source of the division defect in endopeptidase overproducing cells is likely to be the increased competition for substrate between the essential FtsWI synthase in the divisome and aPBPs acting at dispersed sites around the cell to help promote elongation. To our knowledge, a connection between PG cleavage by the endopeptidases and the activity of the Rod system has yet to be demonstrated. Nevertheless, it would be surprising if the activities of the two systems were not at least indirectly linked. We therefore suspect that enhanced Rod system activity is also likely to contribute to the impaired divisome function observed upon endopeptidase overproduction.

In conclusion, our results indicate that PG endopeptidases can influence the division process in addition to promoting cell elongation, highlighting the critical role that controls governing their activity play in the overall cell cycle. So far, the degradation of MepS by NlpI-Prc and the presence of an inhibitory domain in MepM-like endopeptidases are the only known factors affecting endopeptidase activity (Shin et al., 2020). How these systems are modulated during growth or what signals they might respond to remain unclear. Therefore, just as we are only beginning to understand the regulation of divisome activation, much also remains to be learned about the mechanisms controlling cell elongation and how the activity of the two processes are intertwined to promote normal morphogenesis.

EXPERIMENTAL PROCEDURES

Media, bacterial strains and plasmids

As indicated, cells were grown in LB [1% tryptone, 0.5% yeast extract, 0.5% NaCl], LB0N [1% tryptone, 0.5% yeast extract], 0.5xLB0N [0.5% tryptone, 0.25% yeast extract] or minimal M9 medium (Miller, 1972) supplemented with 0.2% casamino acids and 0.2% glucose or arabinose. Unless otherwise indicated, antibiotics were used in LB medium at 25 (chloramphenicol; Cm), 25 (kanamycin; Kan), or 50 (ampicillin; Amp) μg/ml. For M9 medium, 50 μg/ ml Kan and 25 μg/ml Cm were used.

The bacterial strains used in this study are listed in Table S2. All E. coli strains used in the reported experiments are derivatives of MG1655 {Guyer:1981gq}. Plasmids used in this study are listed in in Table S3. Polymerase chain reaction (PCR) was performed using Q5 polymerase (NEB) for cloning purposes and Taq DNA polymerase (NEB) for diagnostic purposes, both according to the manufacturer’s instructions. Unless otherwise indicated, MG1655 chromosomal DNA was used as the template. Plasmid DNA and PCR fragments were purified using the Zyppy plasmid miniprep kit (Zymo Research) or the Qiaquick PCR purification kit (Qiagen), respectively.

ΔSPORftsN::KanR strain construction

The ΔSPORftsN::KanR allele was constructed by replacing the C-terminal SPOR domain from residues 244–319 with the KanR cassette as described previously (Yu et al., 2000; Baba et al., 2006). The KanR cassette was amplified from pKD13 (Datsenko and Wanner, 2000) using the primers 5’-TCGTGCCGCTGACGCGCCAAAACCGACGGCGGAGAAATAAATTCCGGGGATCCGTCGACC-3’ and 5’-GGGCATAATGCAATTATAGATGGGGGGGATTTTGAGGGTTTGTAGGCTGGAGCTGCTTCG-3’. The resulting PCR product was purified and electroporated into strain TB10 as described previously (Bernhardt and de Boer, 2004), and the recombinants were selected at 30°C on LB agar containing 25 μg/ml Kan.

Microscopy and image analyses

Specific growth conditions for each experiment prior to microscopy are described in the figure legends. One microliter of the resulting culture was spotted on 2% agarose pads made using M9 or LB0N medium as indicated and covered with #1.5 coverslips. Images were obtained using a Nikon Ti inverted microscope equipped with a Nikon motorized stage, an Andor Zyla 4.2 Plus sCMOS camera, Lumencore SpectraX LED Illumination, Plan Apo 100x/1.4 Oil Ph3 DM objective lens, and Nikon Elements 4.30 acquisition software. Images in the green channel were taken using a Chroma 49002 filter cube. Images shown in the figures were cropped and adjusted using FIJI software (Schindelin et al., 2012). Automated cell segmentation and identification as well as measurements of cell length were carried out using Oufti (Paintdakhi et al., 2016).

The total cell area was measured by first applying a gaussian blur (sigma = 2), using the Auto Threshold plugin using the default method to segment cells, and then the FIJI Analyze Particles plugin. Normal ZapA-GFP were identified using the Laplacian of Gaussians (LoG) detector in the FIJI Trackmate plugin (Tinevez et al., 2017). The estimated blob diameter was 1.0 μm with threshold 0.5 for ZapA-GFP. The intensity filters for spot detection were adjusted to detect all well-formed rings in the wild-type strain with empty vector control. The same settings were then applied to the remainder of each dataset. Examples of ZapA-GFP rings identified using this method can be found in Figure S2.

Transposon mutagenesis

E. coli TB28 [WT] and DL81 [ΔftsP] were mutagenized with the KanR mariner transposon delivered by conjugation from the donor strain MFDpir/pSC189, which is auxotrophic for diaminopimelic acid (DAP). The donor strain was grown in LB supplemented with 300 μM DAP (LB + DAP) and 50 μg/mL ampicillin, and the recipient strains were grown in LB overnight at 37ºC. The cells were then washed in LB + DAP and concentrated by resuspension in 1/10th the original culture volume of LB + DAP. Equal volumes of donor and recipient were mixed together and 50 μL aliquots were spotted on 0.45 μm filters (Millipore) placed on LB plates. Donor only and recipient only aliquots were also plated in parallel to verify successful selection of exconjugants containing the transposon and counterselection against the donor. The plates were incubated at 37ºC for a one hour mating. Cells were resuspended in LB supplemented with 25 μg/mL Kan, plated on LB0N agar supplemented with 25 μg/mL Kan, and incubated at 30ºC overnight. These matings yielded 1.5–1.6 × 106 colony forming units. Transposon mutant libraries were harvested by scraping the cells from the plates. They were then resuspended in LB0N supplemented with 25 μg/mL Kan, glycerol was added to a final concentration of 16%, and aliquots were frozen at −80ºC.

Transposon insertion sequencing

Transposon insertions were sequenced as previously described (Greene et al., 2018; Lim et al., 2019). Genomic DNA was extracted from the mutant library pellets, fragmented, purified, and poly-C tailed. The transposon-chromosome junctions were amplified in a PCR reaction using the following primers specific to the poly-C tail and transposon:

PolyG-1st-1 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGGG

GGGGGG-3’

pSC189–1st-3 5’-GGCTGACCGCTTCCTCGTGCTTTAC-3’

The transposon junctions were then further amplified and barcoded during a second nested PCR reaction using the NEBNext Multiplex Oligos for Illumina (NEB) and the following primer:

pSC189–2nd-1 5‘-AATGATACGGCGACCACCGAGATCTACACTCTTTCGGGGACTTATCAGCCAACCTG-3’

The PCR products from each library were pooled, run on a 2% agarose gel, and fragments in the 200–500 basepair range were extracted and gel purified. The barcoded libraries were sequenced using a MiSeq reagent kit v3 (150-cycle) (Illumina) using the following custom primer:

BTK30_IL_seq_v2 5’-CTTTCGGGGACTTATCAGCCAACCTGTTA-3’

Sequenced reads were demultiplexed, trimmed, and mapped to the E. coli MG1655 genome. The read counts were normalized to the library with the fewest number of reads and then a Mann-Whitney-U test was used to compare and identify significant differences between the insertion profiles in each library. Genes with at least a 2.8-fold enrichment or depletion and a p-value < 0.05 were defined as significant. Insertion profiles were visualized using the Sanger Artemis Genome Browser.

Supplementary Material

sup 01

ACKNOWLEDGMENTS

The authors would like to thank all members of the Bernhardt and Rudner labs and Piet de Boer for helpful discussions and suggestions. We would also like to thank the Microscopy Resources on the North Quad (MicRoN) core at Harvard Medical School for microscopy support, and Alyson Warr and the Waldor lab for advising us on generating our transposon libraries using pSC189. This work was supported by the National Institutes of Health (AI083365 to T.G.B) and Investigator funds from the Howard Hughes Medical Institute. T.T.T. was supported in part by an NSF pre-doctoral fellowship (DGE1745303) and A.V. was supported in part by an EMBO fellowship (ALTF 89–2019). Our microscope was purchased in part from funds provided by grant S10 RR027344–01 from the National Institutes of Health.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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sup 01
NIHMS1630017-supplement-sup_01.pdf (5.3MB, pdf)

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